Williamsia muralis bacteraemia in a patient with Fanconi anaemia after haematopoietic cell transplantation

Introduction. Williamsia muralis is an environmental bacterium first detected in 1999. Infections with W. muralis isolated have been reported in two elderly patients, and were associated with the surgical intervention of artificial objects. We present a case of bacteraemia caused by W. muralis following haematopoietic cell transplantation (HCT). Case presentation. A 10-year-old Japanese boy presented with fever and the swelling of the left cheek 8 days after HCT for the treatment of Fanconi anaemia. Gram-positive, rod-shaped bacteria were isolated from the blood cultures after 5 days incubation. 16S rRNA sequencing, but not mass spectrometry, identified a strain of W. muralis (1 414 bp, %ID 100 %). The phlegmon did not respond to antimicrobial therapy, but remitted with defervescence after a successful engraftment with teicoplanin and meropenem therapy on day 16 after HCT. The patient experienced recurrence of the bacteraemia, leading to central venous catheter (CVC) line removal. The same strain of W. muralis was isolated from the cultured tip of the CVC. To our knowledge, this is the first reported case of W. muralis bacteraemia and was complicated by CVC infection after HCT. Conclusion. W. muralis bacteraemia developed in an immunocompromised child. Introduction of artificial objects into the body raises a risk of rare infection with slowly growing environmental bacteria.

Limited information is available about human infection with Williamsia spp.because only a few cases of isolated infection have been reported in elderly patients [3].It appeared that W. muralis infection was associated with surgical intervention/prosthetic material.We herein present the first case, to our knowledge, of W. muralis bacteraemia, which occurred in a paediatric patient after haematopoietic cell transplantation (HCT).

CASE PRESENTATION Clinical findings
A 10-year-old Japanese boy with transfusion-dependent Fanconi anaemia underwent HCT.On day 8 after HCT, the patient complained of abdominal pain with vomiting and bloody stool (Fig. 1).His body temperature was 38 °C.The laboratory findings revealed a white blood cell count of 40 cells µl −1 and an elevated C-reactive protein concentration of 2.81 mg µl −1 .For the control of post-transplant febrile neutropenia, cefepime 50 mg kg −1 , twice daily, was administered after the first blood culture from the central venous catheter (CVC).Because of persistent fever for 4 days, the antibacterial agent was switched to meropenem, 100 mg kg −1 per day for 24 days, after the second blood culture.His left cheek became swollen on day 14 after HCT.Enhanced computer tomography findings led to a diagnosis of facial cellulitis (Fig. 2a).

Investigations and diagnosis
On microbiological analysis, Peds Plus (PED) and Plus Anaerobic culture vials (Becton Dickinson; BD) were used in a BD BACTEC FX system.Gram-positive, rod-shaped bacteria were isolated from the first two blood cultures in PED vials after a 5 day incubation period.Both blood culture-positive samples were collected from the CVC.Small colonies were observed after incubation for 48 h at 35 °C on 5 % sheep blood agar plates (Shimazu) in a 5 % CO 2 environment.These colonies appeared as round, pale orange, Gram-positive bacilli that expanded to a diameter of 2-3 mm after 5 days of additional incubation (Fig. 2b).The isolated bacteria were catalase-positive, but negative for Kinyoun's acid-fast staining.However, it was difficult to identifying the Gram-positive bacilli biochemically because of disparate results between VITEK 2 ANC and API Coryne tests.The VITEK2 ANC system identified the bacilli as Corynebacterium urealyticum (% ID: 96 %), while the API Coryne test after 24 h of incubation indicated that the bacilli were Rhodococcus spp.(% ID: 94.7 %).VITEK MS, an automated mass spectrometry using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology, didn't identify the strain either.Because of the failure to determine the strain using these devices and systems, we performed 16S rRNA sequencing with an ABI 3500xL automated DNA sequencer (Applied Biosystems) under the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (MM18-A) [4].The sequence obtained had a continuous stretch of 1 414 bp that exhibited 100 % similarity to W. muralis in a previous report [1].The antimicrobial-drug susceptibility of the isolated colony was determined using the agar plate dilution method.The isolate was incubated on microplates at 35 °C in an aerobic atmosphere for 72 h.The minimum inhibitory concentration of W. muralis was estimated as described by CLSI document M24-A2 [5], and susceptibility to several antibiotics, including meropenem and teicoplanin, was observed (Table 1).

Treatment and outcome
Three days after an additional administration of teicoplanin (initially 10 mg kg −1 every 12 hours for 3 doses, then 10 mg kg −1 once daily for 14 days), fever, C-reactive protein levels and cheek swelling peaked out on day 16 after HCT, coinciding with engraftment (i.e.white blood cells, 1010 cells µl −1 , and neutrophils, 900 cells µl −1 ).A prompt improvement of fever and cellulitis was observed by day 18 after HCT (Fig. 1).The post-transplant management required the persistent insertion of a CVC after HCT.Based on the result that the blood culture from the CVC on day 14 was sterile, antibiotic therapy was completed (23 days for meropenem and 15 days for teicoplanin).However, fever recurred on day 48 after HCT.The blood culture from the CVC was positive again for W. muralis, although the peripheral venous blood cultures were negative on day 48 and day 55 post-transplant.Under the diagnosis of catheter-related bloodstream infection, the CVC was removed.W. muralis was isolated again from the culture of the CVC tip.Meropenem, 100 mg kg −1 per day for 7 days, therapy was terminated 1 week after CVC removal, and no fever recurred thereafter.

DISCUSSION
We have described a case of W. muralis infection in an immunocompromised child who presented recalcitrant bacteraemia complicated by CVC infection.This is, to our knowledge, the first report of W. muralis bacteraemia, because the two previous cases were reported as having isolated infection with W. muralis presenting with endophthalmitis and pneumonia [6,7].
We compared this rare human infection between the current case and the two reported cases (Table 2).W. muralis infection developed after the insertion of artificial objects during surgery, intraocular treatment or central venous nutrition.These findings alert that W. muralis infection occurs when environmental bacteria attach to these artificial objects.The three cases also indicate that W. muralis has a pathogenic potential in immunocompromised hosts.In the reported cases, local lesions developed within a few days after surgery.The first patient without immunodeficiency died of multiple organ failure from pneumonia.The short incubation time of the slowly growing bacteria indicated a property of the strain, otherwise a systemic infection occurring from the greater amount of contamination in the aged patient.Nevertheless, there was no description of the blood cultures in the deceased case.The facial cellulitis in the current case was regrettably not confirmed as bacteraemia origin because no punctures of the affected lesion were performed.Similarly, it was difficult to determine that the preceding bloody diarrhoea was induced by bacteraemia of W. muralis without identification in stool culture.We should also remark on the difficulty of laboratory identification of this organism and the risk of misidentification by commercially available biochemical systems.It is unclear whether the environmental bacteria of W. muralis form biofilms in the host.In this case, there was recalcitrant bacteraemia prior to line removal and positive culture from the line tip.W. muralis was considered to have enough time to form biofilm on the CVC tip during the 28 days after the insertion and during the conditioning phase of HCT.These findings could provide evidence that the environmental bacteria of W. muralis form biofilm on inserted artificial objects.
The clinical features of W. muralis infection share similarities with those of nocardiosis, occasionally causing a localized or disseminated infection in immunocompromised hosts.Nocardia spp.are environmental bacteria belonging to the same family (Nocardiaceae) as W. muralis.Nocardiosis particularly affects the skin, soft tissue and the nervous system through haematogenous dissemination.Unlike Nocardia spp., W. muralis might be susceptible to a wide range of antimicrobial drugs.Because there are no specific criteria for antimicrobial-drug susceptibility testing in Williamsia spp., we used the criteria for Nocardia spp., as described [5].The strain this patient contracted exhibited a wide spectrum of antimicrobial susceptibility.Further identification of cases of infection is awaited to determine an effective strategy of antimicrobial drugs and treatment duration.

Conclusion
We report the first case, to our knowledge, of W. muralis bacteraemia in an immunocompromised host.The isolated or systemic infection with W. muralis suggested similarities with nocardial infections occurring after the insertion of artificial objects.

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Find out more and submit your article at microbiologyresearch.org I'd suggest also mentioning in the main introduction (Section 4) that previous cases of W. muralis infection have been associated with intervention / prosthetic material.
In accordance with the reviewer's comment, we have moved the sentence about previous cases and added the comment for artificial objects in the abstract (Section 2, p.1, lines 25-27) and the main introduction (Section 4, p.2, lines 56-58).
b. Abstract (Section 2) Case presentation -please make it clear that there was recurrence of bacteraemia, leading to line removal and positive line tip culture.
Accordingly, we have changed the abstract on p.2, lines 35-37.
c. Figure 1 and 2 are excellent and table 1 with AST is very clear.
We strongly appreciate the reviewer's comment on this point.
3. How the style and organization of the paper communicates and represents key findings a. Abstract (Section 2) Case presentation -stating "bacteraemia was determined to arise from the catheter-related bloodstream infection" -I don't think it's possible to determine if this is a primary CRBSI or secondary to facial cellulitis.I'd suggest rewording this sentence to reflect it's the first reported case of W. muralis bacteramia and was complicated by CVC infection.
The reviewer's comment is correct.We have changed the sentence on p.2, line 38.
b. Introduction (Section 4) -last statement "findings provide evidence that W. muralis forms biofilm on the inserted artificial objects" -this is speculation without any previous reference to the presence of CVC in the introduction.Please consider moving this to the discussion section (Section 7) and add supporting comments, such as recalcitrant bacteramia prior to line removal and positive culture from line tip.
Accordingly, we have moved the sentence in the introduction to the discussion and added the supporting comments.(p.7, lines 149-154).

Literature analysis or discussion
a. Table 2 is good -clearly presents previous cases of W. muralis infection.
We thank the reviewer for this comment.
b. Discussion (Section 7) -"facial cellulitis with bacteraemia originating from the CVC" -this implies CVC is the primary source of bacteraemia which I don't think is possible to confirm / refute.As before, I'd suggest rewording this.
The reviewer's comment is correct as before.We have changed the sentence on p.7, line 130.
c.In Introduction (Section 4) and Figure 1 -reference made to bloody diarrhoea but no further comment in the manuscript.Please comment in the Discussion (Section 7) if bloody diarrhoea was thought to be related or unrelated to bacteraemia.
Accordingly, we have added the comment about the relationship between bloody diarrhea and bacteremia on p.7, lines 145-146.
As well as facial cellulitis, it was difficult to confirm / refute their relationship without the positive result of the stool culture.
d. Discussion (Section 7)-I'd suggest a comment about difficulties in laboratory identification of this organism and risk of misidentification with commercial biochemical systems.I think this is a key message from this paper.
We thank the reviewer for this comment.We have added the above message on p.7, lines 146-148.

Any other relevant comments
a. Abstract (Section 2) -"blood cultures after five day's incubation" -remove apostrophe.
Thank you, this error has been corrected.
b. Discussion (Section 7) -"The first oldest patient without immunodeficiency" should be reworded for clarity -suggest removing "oldest".
c. Discussion (Section 7) -"... there was no description of the positive blood culture" -this implies there was a positive blood culture, consider rewording to clarify if any mention of blood cultures in the paper.
We agree that this point requires clarification, we have removed " positive ".
d. Discussion (Section 7) -consider rewording to clarify what is meant by a "blood-born lesion" -source of bacteraemia or metastatic site of infection?
Comments: Interesting case.I really like the Figure 1 -it makes the timeline very clear.The only minor adjustments I would suggest are with the language -in general this is good but there is the odd sentence where the choice of words does not make clear sense and could do with proofreading e.g.line 131-133 "These suggest a precaution that W. muralis infection occurs when environmental bacteria attach to these artificial objects."-I am not sure that the word precaution is the correct one in this context.

Please rate the quality of the presentation and structure of the manuscript Very good
To what extent are the conclusions supported by the data?Case presentation -previous reports of W. muralis infection should be moved to the introductory section above this and should state these were associated with prosthetic material / intervention to support comment about introduction of artificial objects in the abstract conclusion section.I'd suggest also mentioning in the main introduction (Section 4) that previous cases of W. muralis infection have been associated with intervention / prosthetic material.b. Abstract (Section 2) Case presentation -please make it clear that there was recurrence of bacteraemia, leading to line removal and positive line tip culture.c. Figure 1 and 2 are excellent and table 1 with AST is very clear.3. How the style and organization of the paper communicates and represents key findings a. Abstract (Section 2) Case presentation -stating "bacteraemia was determined to arise from the catheter-related bloodstream infection" -I don't think it's possible to determine if this is a primary CRBSI or secondary to facial cellulitis.I'd suggest rewording this sentence to reflect it's the first reported case of W. muralis bacteramia and was complicated by CVC infection.b.Introduction (Section 4) -last statement "findings provide evidence that W. muralis forms biofilm on the inserted artificial objects" -this is speculation without any previous reference to the presence of CVC in the introduction.Please consider moving this to the discussion section (Section 7) and add supporting comments, such as recalcitrant bacteramia prior to line removal and positive culture from line tip.4. Literature analysis or discussion a.Table 2 is good -clearly presents previous cases of W. muralis infection.b. Discussion (Section 7) -"facial cellulitis with bacteraemia originating from the CVC" -this implies CVC is the primary source of bacteraemia which I don't think is possible to confirm / refute.As before, I'd suggest rewording this.c.In Introduction (Section 4) and Figure 1 -reference made to bloody diarrhoea but no further comment in the manuscript.Please comment in the Discussion (Section 7) if bloody diarrhoea was thought to be related or unrelated to bacteraemia.d. Discussion (Section 7)-I'd suggest a comment about difficulties in laboratory identification of this organism and risk of misidentification with commercial biochemical systems.I think this is a key message from this paper.5. Any other relevant comments a. Abstract (Section 2) -"blood cultures after five day's incubation" -remove apostrophe.b. Discussion (Section 7) -"The first oldest patient without immunodeficiency" should be reworded for clarity -suggest removing "oldest".c. Discussion (Section 7) -"... there was no description of the positive blood culture" -this implies there was a positive blood culture, consider rewording to clarify if any mention of blood cultures in the paper.d. Discussion (Section 7) -consider rewording to clarify what is meant by a "blood-born lesion" -source of bacteraemia or metastatic site of infection?

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No

Fig. 2 .
Fig. 2. (a).Facial cellulitis on the patient's left cheek (yellow circles).(b).Blood culture results over time.Gram-positive, rod-shaped bacteria were isolated from the blood culture samples after 5 days of incubation.Small colonies were observed after 2 days of incubation on 10 % sheep blood agar plates (left picture).The colonies were round, pale orange, Gram-positive bacilli, and their diameter expanded to 2-3 mm after 5 days of additional incubation (middle and right pictures).

Table 2 .
Clinical presentation of three patients who developed W.
Thank you -interesting case. 1. Description of the case(s) a. Investigations and diagnosis (Section 5) -please clarify if the blood cultures were processed in an automated continuously monitored commercial blood culture machine and manufacturer or is this a terminal subculture?b.Investigations and diagnosis (Section 5)-please clarify if the positive blood cultures are peripheral / CVC and if aerobic / anaerobic bottles or separate sets.c.Investigations and diagnosis (Section 5) -please clarify if 16S PCR was done by Sanger sequencing / platform used / if external laboratory.d.Investigations and diagnosis (Section 5) -please expand on VITEK MS to make clear it's a mass spectroscopy, MALDI-TOF system.e. Investigations and diagnosis (Section 5)please clarify if agar plate dilution microplates are prepared in house or if commercial and what media is used.f.Treatment and outcome (Section 5)-how was it determined the same strain of W. muralis isolated again?This statement should be supported or simply state that W. muralis was isolated again.2. Presentation of results a. Abstract (Section 2)